Cloning And Expression Of Human CXCR4,Arresten Gene And Biological Activity Analysis Of Arresten

Chemokines and chemokine receptors are important regulatory factors in tumor behaviors. chemokine actions lead to activation of G proteins and phospholipase C, and stimulate tumor growth and metastases by increasing survival, proliferation, migration, adhesion to extracellular matrix of cancer cells.CXCR4 and its natural ligand SDF-1 are widely expressed in cancer cells and play a key role in metastasis-related behaviors of cancer cells, such as attachment, spreading, migration and invasion. Antibodies against CXCR4 have been thought to be useful for the research into CXCR4 functions and treatment of cancer. To express recombinant CXCR4 and prepare its antibodies, The recombinant plasmid pGEX-4T1-CXCR4 was constructed and transformed into E.coli BL21 (DE3) , and the target gene was expressed under induction of IPTG. Expression of CXCR4 was found to induce cytotoxic effect in transfected E.coli cells. However, the CXCR4 N-terminal extracelluar domain peptide (1-42aa), involved in ligand binding, was successfully expressed as GST fusion proteins in E.coli. Recombinant CXCR4 N-terminal was obtained with a purity of 80% by sepharose 4B affinity chromatography and ammonium sulfate precipitation. These work lays a foundation for selection of anti-CXCR4 antibodies.Angiogenesis is crucial for the growth, invasion and metastases of malignant tumors. Accumulating evidences indicate that antiangiogenesis therapies can suppress proliferation and migration of tumor cells effectively. Antiangiogenesis as a strategy for treatment of cancer has been vigorously pursued.arresten is a newly found endogenous antiangiogenic peptide derived from type IV collagen a1-chain C-terminal noncollagenous domain 1 (NC1). In animal models, arresten can suppress tumor growth and invasion effectively. To further investigate antiangiogenic effect of arresten, recombinant arresten was expressed in E.coli BL21(DE3). The target fusion protein was found to exist mainly in the pellet of broken cells in the form of inclusion body. The soluble recombinant arresten was purified by sepharose 4B affinity chromatography. Biological activity of recombinant arresten was analyzed by the technique of chicken chorioallantoic membrane (CAM). The experimental results showed that purified soluble recombinant arresten could suppress proliferation of blood vessel on CAM.